Protein kinase C (See Drosophila PKC) is activated at the nuclear membrane in response to a variety of mitogenic
stimuli. In human leukemic cells, the beta II PKC isotype is selectively translocated and activated at
the nucleus. The nuclear envelope component lamin B1 is a major substrate for
nuclear PKC both in whole cells and in vitro. Using highly purified human beta II PKC and isolated
nuclear envelopes from the human promyelocytic (HL60) leukemia cell line,
the major sites for beta II PKC-mediated lamin B phosphorylation have been determined. Two
major sites of PKC-mediated phosphorylation, Ser395 and Ser405, have been identified. These sites lie
within the carboxyl-terminal domain of lamin B immediately adjacent to the central alpha-helical rod
domain. Functionally, beta II PKC-mediated phosphorylation of these sites leads to the time-dependent
solubilization of lamin B, indicative of mitotic nuclear envelope breakdown in vitro. beta II
PKC-mediated lamin B phosphorylation is inhibited by (1) a monoclonal antibody directed against the
active site of PKC, (2) a PKC pseudosubstrate inhibitor peptide, and (3) a PKC peptide substrate. Two
observations indicate that PKC-mediated lamin B phosphorylation and solubilization are due to direct
phosphorylation of lamin B by PKC rather than indirect activation of a cdc2 kinase. Neither
immunodepletion with p13suc1 Sepharose beads nor the presence of a p34cdc2 kinase peptide
substrate had any effect on PKC-mediated lamin B phosphorylation. Therefore, it is concluded that beta
II PKC represents a physiologically relevant lamin kinase that can directly modulate nuclear lamina
structure in vitro. Nuclear beta II PKC, like p34cdc2 kinase, may function to regulate nuclear lamina
structural stability during cell cycle (Hocevar, 1993).

Protein kinase C (PKC) is activated at the nucleus during the G2 phase of cell cycle, where it is
required for mitosis. However, the mechanisms controlling cell cycle-dependent activation of nuclear
PKC are not known. Nuclear levels of the major physiologic PKC activator
diacylglycerol (DAG) fluctuate during cell cycle. Specifically, nuclear DAG levels in G2/M phase cells
are 2.5 to 3 times higher than in G1 phase cells. In synchronized cells, nuclear DAG levels rise to a peak
coincident with the G2/M phase transition and return to basal levels in G1 phase cells. This increase in
DAG level is sufficient to stimulate betaII PKC-mediated phosphorylation of its mitotic nuclear
envelope substrate lamin B in vitro. Isolated nuclei from G2 phase cells contain an active phospholipase
activity capable of generating DAG in vitro. Nuclear phospholipase activity is inhibited by the selective
phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor
1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and neomycin sulfate, but not by the
phosphatidylcholine-PLC selective inhibitor D609 or inhibitors of phospholipase D-mediated DAG
generation. Treatment of synchronized cells with
1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine leads to decreased nuclear PI-PLC activity and
cell cycle blockade in the G2 phase, suggesting a role for nuclear PI-PLC in the G2/M phase transition.
These data are consistent with the hypothesis that nuclear PI-PLC generates DAG to activate nuclear
betaII PKC, whose activity is required for mitosis (Sun, 1997).

Disassembly of the sperm nuclear envelope at fertilization is one of the earliest events in the development of the male
pronucleus. Nuclear lamina disassembly in interphase sea urchin egg cytosol is a result of lamin B
phosphorylation mediated by protein kinase C (PKC). Lamin B of permeabilized sea urchin sperm nuclei incubated in fertilized
egg G1 phase cytosolic extract is phosphorylated within 1 min of incubation and solubilized prior to sperm chromatin
decondensation. Phosphorylation is Ca2+-dependent. It is reversibly inhibited by the PKC-specific inhibitor chelerythrine, a
PKC pseudosubstrate inhibitor peptide, and a PKC substrate peptide, but not by inhibitors of PKA, p34(cdc2) or calmodulin
kinase II. Phosphorylation is inhibited by immunodepletion of cytosolic PKC and restored by addition of purified rat brain
PKC. Sperm lamin B is a substrate for rat brain PKC in vitro, resulting in lamin B solubilization. Two-dimensional
phosphopeptide maps of lamin B phosphorylated by the cytosolic kinase and by purified rat PKC are virtually identical. These
data suggest that PKC is the major kinase required for interphase disassembly of the sperm lamina (Collas, 1997).

At the end of mitosis, the nuclear lamins assemble to form the nuclear lamina during nuclear envelope formation in daughter cells. A- and B-type nuclear lamins have been fused to the green fluorescent protein to study this process in living cells. The results reveal that the A- and B-type lamins exhibit different pathways of assembly. In the early stages of mitosis, both lamins are distributed throughout the
cytoplasm in a diffusible (nonpolymerized) state. During the anaphase-telophase transition, lamin B1 begins to become concentrated at the surface of the chromosomes. As the chromosomes reach the spindle poles, virtually all of the detectable lamin B1 has accumulated at their surfaces. Subsequently, this lamin rapidly encloses the entire perimeter of the region containing decondensing chromosomes in each daughter cell. By this time, lamin B1 has assembled into a relatively stable polymer. In
contrast, the association of lamin A with the nucleus begins only after the major components of the nuclear envelope including pore complexes are assembled in daughter cells. Initially, lamin A is found in an unpolymerized state throughout the nucleoplasm of daughter cell nuclei in early G1 and only gradually becomes incorporated into the peripheral lamina during the first few hours of this stage of the cell cycle. In later stages of G1, Both green fluorescent protein lamins A and B1 appear to form higher order polymers throughout interphase nuclei (Moir, 2000).

The differences in lamins A and B1 distributions may reflect their different interactions with other nuclear envelope components. For example, it is known that B-type lamins are associated with nuclear envelope-derived membrane vesicles in mitotic cells. Therefore, lamin B1 would be expected to be localized peripherally during the early stages of assembly as part of the forming nuclear membrane. Furthermore, LAPs
probably mediate the interactions of lamins with membranes. The LAP family includes LAP2alpha and ß, emerin and MAN1. In particular, LAP2 has been implicated in the regulation of the early stages of nuclear assembly and also in the growth of the nucleus during G1. Different fragments of LAP2 can inhibit either nuclear assembly or
nuclear growth, perhaps reflecting the binding of this protein to chromatin or lamins. LAP2alpha, a non-membrane-bound isoform, colocalizes with A-type lamins during nuclear formation and may specifically regulate the assembly of lamin A.
Since LAP2ß may interact primarily with lamin B isoforms, the distributions of lamins A and B1 may reflect interactions with different forms of LAP2
during nuclear assembly (Moir, 2000).

The different distributions of lamins A and B1 may also be due to posttranslational modifications. For example, lamins A and B are isoprenylated at a conserved COOH-terminal cysteine. The isoprenyl group remains on lamin B throughout the cell cycle, but it is rapidly removed from lamin A by an endoprotease. The mutation of the cysteine residue prevents isoprenylation and results in an exclusively nucleoplasmic distribution of lamin A. However, other experiments using inhibitors of isoprenylation suggest that lamin A incorporation into the lamina is not affected by the inhibition of this posttranslational event. In these studies, it is most likely that the majority of the lamin A that is observed
localizing to the nucleus immediately after mitosis is synthesized in the previous cell cycle and therefore would not be isoprenylated as a
consequence of the proteolytic cleavage step. It is possible, therefore, that as new, isoprenylated lamin A is synthesized during G1, it interacts, perhaps by forming
dimers or tetramers, with the nucleoplasmic lamin A synthesized in the previous cell cycle, resulting in the targeting of both populations to the envelope (Moir, 2000).

Lamins and the building of a nuclear envelope

Xenopus oocytes, eggs, and early embryos contain lamins LII and LIII:
portions of each are associated with distinct egg vesicle populations. A
lamin similar or identical to the B-type lamin LI is also present in oocyte nuclei and in egg extracts. The three B-type lamins have been quantified per oocyte nucleus, with relative ratios of
LI:LIII = 1:100, and LII:LIII = 1:10. Similar to lamin LII, 5-15% of lamin LI is associated with egg
membranes in a biochemically stable manner. Egg vesicles absorbed with lamin isoform-specific
antibodies to magnetic beads indicate that lamin LI-associated egg membranes are of heterogenous
morphology, and are independent from the lamin LII and LIII vesicle populations. Compared to other
nuclear envelope proteins, the synthesis of lamin LI protein is specifically elevated during meiotic
maturation, resulting in a 4- to 12-fold higher amount of lamin LI in eggs than is present in oocyte
nuclei. Lamins LI, LII, and LIII are
associated with the nuclear envelope formed on demembranated sperm when added to activated egg
extract. These results strongly suggest that three different lamin-associated vesicle populations are
involved in the formation of a nuclear envelope in egg extracts (Lourim, 1996).

Xenopus egg extracts, which assemble replication competent nuclei in vitro, were depleted of lamin B3
using a specific monoclonal antibody linked to paramagnetic beads. After depletion the extracts were
still capable of assembling nuclei around demembranated sperm heads. Most nuclei assembled in lamin B3-depleted
extracts have continuous nuclear envelopes and well formed nuclear pores. However, several
consistent differences were observed. Most nuclei are small and only attain diameters which are
half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the
inner aspect of the nuclear envelope, appear on its outer surface. Finally, the assembly of nuclear
pores is slower in lamin B3-depleted extracts, indicating a slower overall rate of nuclear envelope
assembly. Since nuclear envelope assembly is mostly normal but
slow in these nuclei, the lamin content of 'depleted' extracts was investigated. While lamin B3 is
recovered efficiently from cytosolic and membrane fractions, a second minor lamin
isoform, which has characteristics similar to those of the somatic lamin B2, remained in the extract.
Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B2
does not co-precipitate with lamin B3 during immunodepletion experiments, several protein species do
specifically associate with lamin B3 on paramagnetic immunobeads. The major protein species
associated with lamin B3 migrates on gels with molecular masses of 102 kDa and 57 kDa, respectively. The mobility of the 102 kDa
protein is identical to the mobility of a major nuclear matrix protein, indicating a specific association
between lamin B3 and other nuclear matrix proteins. Nuclei assembled in lamin B3-depleted extracts
do not assemble a lamina and fail to initiate
semi-conservative DNA replication. However, by reinoculating depleted extracts with purified lamin
B3, nuclear lamina assembly and DNA replication can both be rescued. Thus it seems likely that the
inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence
of a failure to assemble a lamina (Goldberg, 1995).

The fate of several integral membrane proteins of the nuclear envelope has been examined during
mitosis in cultured mammalian cells to determine whether nuclear membrane proteins are present in a
vesicle population distinct from bulk ER membranes after mitotic nuclear envelope disassembly or are
dispersed throughout the ER. The localization of two inner nuclear membrane proteins (lamina associated polypeptides 1
and 2 [LAP1 and LAP2]) and a nuclear pore membrane protein (gp210) was compared to the distribution of bulk ER
membranes. The three nuclear envelope markers become completely
dispersed throughout ER membranes during mitosis.
LAP1 is found in most membranes containing ER markers. Together, these findings indicate that nuclear membranes lose their identity as a subcompartment
of the ER during mitosis. Nuclear lamins begin to reassemble around chromosomes at
the end of mitosis at the same time as LAP1 and LAP2. It is thought that reassembly of the nuclear
envelope at the end of mitosis involves sorting of integral membrane proteins to chromosome surfaces
by binding interactions with lamins and chromatin (Yang, 1997a).

Nuclear envelope disassembly in mammalian cells has been studied using morphological methods. The first
signs of nuclear lamina depolymerization become evident in early prophase as A-type lamins start
dissociating from the nuclear lamina and diffuse into the nucleoplasm. While B-type lamins are still
associated with the inner nuclear membrane, two symmetrical indentations develop on antidiametric
sites of the nuclear envelope. These indentations accommodate the sister centrosomes and associated
astral microtubules. At mid- to late-prophase, elongating microtubules apparently push on the nuclear
surface and eventually penetrate the nucleus. At this point the nuclear envelope becomes freely
permeable to large ligands, as indicated by experiments with digitonin-treated cells and by the massive
release of solubilized A-type lamins into the cytoplasm. At the prophase/prometaphase transition, the
B-type lamina is fragmented, but 'islands' of lamin B polymer can still be discerned on the tips of
congressing chromosomes. At metaphase, the lamin B polymer breaks down into small pieces,
which tend to concentrate in the area of the mitotic spindle. Nuclear envelope breakdown is not
prevented when the microtubules are depolymerized by nocodazole; however, the mode of nuclear
lamina fragmentation in the absence of microtubules is markedly different from the normal one and
involves multiple raffles and gaps, which develop rapidly along the entire surface of the nuclear
envelope. These data suggest that nuclear envelope disassembly is a stepwise process in which the
microtubules play an important part (Georgatos, 1997).

Interaction of lamins with chromatin

Chromatin in eukaryotic nuclei is thought to be partitioned into functional loop domains that are
generated by the binding of defined DNA sequences, named MARs (matrix attachment regions), to the
nuclear matrix. B-type lamins have been identified as MAR-binding matrix components. A-type lamins and the structurally related proteins
desmin and NuMA also specifically bind MARs in vitro. The interaction between MARs
and lamin polymers is saturable, of high affinity, and
evolutionarily conserved. Competition studies revealed the existence of two different types of
interaction related to different structural features of MARs: one involving the minor groove of
double-stranded MAR DNA and one involving single-stranded regions. Similar results habe been obtained for
the interaction of MARs with intact nuclear matrices from rat liver. A model is discussed in which the interaction of
nuclear matrix proteins with single-stranded MAR regions serves to stabilize the transcriptionally active
state of chromatin (Luderus, 1994).

Previous studies have shown that nuclear lamins
bind chromatin directly. A chromatin binding site has been localized to the carboxyl-terminal tail
domains of both A- and B-type mammalian lamins. Recombinant fusion proteins containing the tail
domains of mammalian lamins C, B1, and B2 were analyzed for their ability to associate with rat liver
chromatin fragments. All three lamin tails
specifically bind to chromatin with apparent KdS of 120-300 nM. The chromatin binding region of the lamin C tail maps to amino acids 396-430, a
segment immediately adjacent to the rod domain. Core histones constitute the principal chromatin binding component for the lamin C tail.
Through cooperativity, this lamin-histone interaction could be involved in specifying the high avidity
attachment of chromatin to the nuclear envelope in vivo (Taniura, 1995).

Three B-type lamin isoforms present in the nuclei of mature Xenopus laevis oocytes, and in cell-free egg extracts, have been identified and quantitated . Because Xenopus egg extracts are frequently used to analyze nuclear envelope assembly and lamina functions, it was imperative that the polymerization and chromatin-binding properties of the endogenous B-type egg lamins be investigated. While soluble B-type lamins bind to chromatin, the polymerization of egg lamins does not require membranes or chromatin. Lamin assembly is enhanced by the addition of glycogen/glucose, or by the depletion of ATP from the extract. Moreover, the polymerization of egg cytosol lamins and their binding to demembranated sperm or chromatin assembled from naked lambda-DNA is inhibited by an ATP regeneration system. These ATP-dependent inhibitory activities can be overcome by the coaddition of glycogen to egg cytosol. Glycogen does not alter ATP levels during cytosol incubation, but rather, as glycogen-enhanced lamin polymerization is inhibited by okadaic acid, it is concluded that glycogen activates protein phosphatases. Because protein phosphatase 1 (PP1) is the only phosphatase known to be specifically regulated by glycogen these data indicate that PP1 is involved in lamin polymerization. The results show that ATP and glycogen effect lamin polymerization and chromatin binding by separate and opposing mechanisms (Lourim, 1999).

Lamin and DNA replication

A cDNA encoding Xlamin B1 was cloned from a whole ovary mRNA by RT-PCR. GST-lamin fusion
constructs were generated. Two expression constructs were made, the first, termed delta 2+ lacks sequences
encoding the amino-terminal 'head domain' of lamin B1 but includes sequences encoding the nuclear
localization signal sequence (NLS). The second expression construct, termed delta 2-, lacks
sequences encoding the amino-terminal 'head domain' as well as sequences encoding the NLS.
Purified tuncated proteins, when added to egg extracts prior to sperm
pronuclear assembly, form hetero-oligomers with the endogenous lamin B3. The delta 2+ fusion
protein prevents nuclear lamina assembly but not nuclear membrane assembly. The resulting nuclei
are small, do not assemble replication centers and fail to
initiate DNA replication. When the delta 2- fusion protein is added to egg extracts prior to sperm
pronuclear assembly, lamina assembly is delayed but not prevented. The resulting nuclei, although
small, do form replication centers and initiate DNA replication. When
added to egg extracts after sperm pronuclear assembly is completed, delta 2+, but not delta 2-,
enter the pre-formed nuclei causing lamina disassembly. However, the disassembly of the lamina by
delta 2+ does not result in the disruption of replication centers and indeed these centers remain
functional. These results are consistent with the hypothesis that lamina assembly precedes and is
required for the formation of replication centers but does not support those centers directly (Ellis, 1997).

Transcriptional regulation of Lamins

The expression of lamins A, B1, and C was examined in human tissues and cancer cell lines and
the function of the lamin A/C and B1 gene promoters were examined in transfected cells. Lamin A/C mRNA and protein were not detectable in some human
cell lines, whereas lamin B1 was always present. Sequencing of approximately 2.6 kb of the lamin A/C
and 1.6 kb of the lamin B1 genes 5' to the translation initiation sites showed that they did not contain
typical TATA boxes near the transcription start sites. The lamin B1 and A/C proximal promoter
regions are transcribed in transfected HeLa, Raji, and NT2/D1 cell lines even if the cells did not
contain detectable endogenous lamin A/C mRNA or protein. These results show that
transcriptional regulatory elements in the promoters of the
human nuclear lamin A/C and B1 genes do not control their cell type-specific expression in culture
lines, as is the case with most cytoplasmic intermediate filament genes (Lin, 1997).